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US6258533B1 - Iterative and regenerative DNA sequencing method - Google Patents

Iterative and regenerative DNA sequencing method Download PDF

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US6258533B1
US6258533B1 US09/035,183 US3518398A US6258533B1 US 6258533 B1 US6258533 B1 US 6258533B1 US 3518398 A US3518398 A US 3518398A US 6258533 B1 US6258533 B1 US 6258533B1
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dna
nucleotide
adaptor
nucleic acid
sequencing
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Douglas H. Jones
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University of Iowa Research Foundation UIRF
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Priority to PCT/US1999/004883 priority patent/WO1999045153A2/fr
Priority to AU30695/99A priority patent/AU3069599A/en
Assigned to IOWA RESEARCH FOUNDATION, UNIVERSITY OF reassignment IOWA RESEARCH FOUNDATION, UNIVERSITY OF ASSIGNMENT OF ASSIGNORS INTEREST (SEE DOCUMENT FOR DETAILS). Assignors: JONES, DOUGLAS H.
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6869—Methods for sequencing
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6813—Hybridisation assays
    • C12Q1/6827—Hybridisation assays for detection of mutation or polymorphism
    • C12Q1/683—Hybridisation assays for detection of mutation or polymorphism involving restriction enzymes, e.g. restriction fragment length polymorphism [RFLP]
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6844—Nucleic acid amplification reactions
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6844—Nucleic acid amplification reactions
    • C12Q1/6853—Nucleic acid amplification reactions using modified primers or templates
    • C12Q1/6855—Ligating adaptors
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6869—Methods for sequencing
    • C12Q1/6874—Methods for sequencing involving nucleic acid arrays, e.g. sequencing by hybridisation

Definitions

  • the invention features a method for identifying a first nucleotide n and a second nucleotide n+x in a double stranded nucleic acid segment.
  • the method includes (a) digesting the double stranded nucleic acid segment with a restriction enzyme to produce a double stranded molecule having a single stranded overhang sequence corresponding to an enzyme cut site; (b) providing an adaptor having a cycle identification tag, a restriction enzyme recognition domain, a sequence identification region; (c) hybridizing the adaptor to the double stranded nucleic acid having the single-stranded overhang sequence to form a ligated molecule; (d) amplifying the ligated molecule from step (c) with a labeled primer specific for the cycle identification tag, restriction enzyme recognition domain and a portion of the sequence identification region of the adaptor; (e) identifying the nucleotide n by identifying the primer incorporated into the amplification product; and (f) repeating step (a)
  • Yet another aspect of the invention pertains to a method for sequencing an interval within a double stranded nucleic acid segment by identifying a first nucleotide n and a second nucleotide n+x in a plurality of staggered double stranded molecules produced from the double stranded nucleic acid segment.
  • the method proceeds by reducing length of each strand at the end of the DNA segment with the overhang template by >1 nucleotide to produce a corresponding set of shorter DNA segments each with an overhang template.
  • the step of reducing is performed by removing a block of nucleotides, so that each shorter DNA segment with an overhang template is a known subinterval of a previous DNA segment with overhang.
  • the steps of treating, reading, regenerating and reducing the length of the strands of the DNA segment at each holder by a number of n ⁇ 1 nucleotides are iteratively performed as automated process steps to produce nested and progressively shorter DNA segment ends and to sequence the plurality of DNA segments immobilized at the array of sample holders in situ.
  • the invention further contemplates an automated instrument for effectively performing the sequencing, wherein a stage carries the support on a device equipped for providing the respective buffers, solutions and reagents, for stepping or positioning the array for reading, and in some embodiments robotic manipulation for sample transfer, and heating for amplification, e.g., treating at least a portion of material at each sample holder with a primer and heat cycling to regenerate material at the respective sample holders.
  • the stage may be rotatable, spinning to cause fluid provided at a central position to centrifugally flow across the array to alter material immobilized in the sample holders.
  • the stage holds plural support arrays, and may operate robotically to transfer material from the sites of one support array to the sites of another support array, so that all the samples on one support may undergo one set of process steps in common (e.g., washing, digestion, labeling) while those on the other support undergo another (e.g., heating/amplification or scintillation reading).
  • process steps e.g., washing, digestion, labeling
  • FIG. 5 is a photograph depicting the size of the initial template precursor and of subsequent template precursors following each of five iterative sequencing simulation cycles consisting of FokI digestion, adaptor ligation, fill-in with ddNTPs, and PCR amplification, run on a 12% denaturing acrylamide gel.
  • FIG. 6 is a schematic diagram which illustrates the removal of primer encoded sequence from a PCR product by amplification with a primer encoding a DpnI recognition domain, which requires a methylated nucleotide, followed by cutting DpnI.
  • the primer sequences are underlined.
  • the primer encoding the DpnI recognition domain had two mismatches with the original PCR template, and the two mismatched nucleotides are depicted in bold.
  • the present invention pertains to an iterative and regenerative method for sequencing DNA that exploits the separation of the restriction enzyme recognition and cleavage domains in class-IIS restriction endonucleases, as well as adaptor ligation, to generate a series of sequencing templates that are separated from each other by a discrete interval.
  • These sequencing templates constitute a set of single-strand overhangs that can then be sequenced by template-directed ligation, template-directed polymerization, or by stringent hybridization of oligonucleotides or oligonucleotide analogs.
  • nucleotide n is selected by digesting a given double stranded nucleic acid segment with a restriction enzyme, e.g., a class IIS restriction endonuclease, to generate a 5′ or a 3′ single stranded overhang sequence corresponding to the cut site, and n is the first or the last unpaired nucleotide in the overhang sequence.
  • a restriction enzyme e.g., a class IIS restriction endonuclease
  • a recognition domain with this characteristic allows one to use primer extension during the polymerase chain reaction (PCR) to hemi-methylate each of the recognition domains except for that recognition domain encoded by the amplifying primer. This is accomplished by using a methylated nucleotide that is not present in the recognition domain sequence that is antisense to the primer encoding this domain. By using a methylated dNTP that does not lie in the strand antisense to the recognition domain encoded in the amplifying primer, all the recognition domains in the PCR product are methylated except the recognition domain that is encoded by the amplifying primer. This strategy hemi-methylates each recognition domain in the PCR product except the primer-encoded recognition domain. This approach has been applied using a recognition domain for a class II-S restriction endonuclease, to generate recombinant constructs (Padgett K A, J A Sorge Gene 1996; 168:31-35).
  • An adaptor of the invention is a double stranded or a single stranded polynucleotide having one or more of a cycle identification tag, a restriction enzyme recognition domain and a sequence identification region.
  • the adaptor may also include a detectable label, which in the particular embodiment of FIG. 1 is illustrated at the end opposite of the sequence identification region.
  • Uracil DNA Glycosylase is used to cleave the N-glycosylic bond between the deoxyribose moiety and uracil, resulting in an abasic site (Varshney U, T Hutcheon, J H van de Sande, J Biol Chem 1988; 263:7776-7784).
  • FokI generates a four nucleotide long 5′ overhang positioned nine nucleotides away from one side of the recognition domain, so that sequencing can be carried out in intervals of nine nucleotides.
  • Fok I digestion cleaves both strands of the double-stranded DNA, generating a DNA template with a 5′ overhang sequence. The bound template is washed to remove the cleaved ends.
  • Step 2 the 5′ overhang sequence mediates ligation to one of four adaptors.
  • These adaptors contain the sequence for the recognition domain for Fok I and have an adjacent four nucleotide long and phosphorylated 5′ overhang consisting of three nucleotides with 4-fold degeneracy and a 5′ terminus with one of the four normal nucleotides. Since the four adaptors each have three degenerate nucleotides and four distinct 5′ terminal nucleotides, there are 256 distinct sequences. The adaptors shown arc double-stranded, because this increases the ligation efficiency, probably due to stacking interactions (Lin S-B, K R Blake, P S Miller, Biochemistry 1989; 28:1054-1061). In this embodiment of the method there is one ligation reaction during each sequencing cycle.
  • this polymerization may be performed with concurrent hemi-methylation of the adaptor encoded recognition domain for the class-IIS endonuclease using the polymerase extension in the presence of a methylated nucleotide (when sequencing with a class-IIS restriction endonuclease that recognizes a hemi-methylated recognition domain; also, if the ligated upper-strand's recognition domain sequence were methylated, both strands of the recognition domain would be methylated using this method).
  • the template precursors can be bound to a silicon chip or contained in a matrix of chambers, so that cycles of adaptor ligation, template-directed DNA polymerization for amplification or sequencing, and cutting can be carried out on numerous templates in parallel.
  • each of the DNA segments which are to be analyzed which may, for example, be PCR products or vector inserts, is immobilized so that it resides at a unique address on the chip or support 10 , and several hundred to thousands of DNA segments are distributed on the chip. They simultaneously undergo a series of incubations that result in the accumulation of sequence information.
  • a reagent may be delivered, for example, by a robotically carried comb or pipette array, or preferably by bulk or flow-through addition of the reagent. Separate reagents in their respective buffers are represented by the jar in the left hand portion of the diagram and these are passed to the support array 10 by automated control in the order for performing the sequencing chemistry described herein.
  • the method preferably includes a regeneration step.
  • a regeneration step Illustratively, following the adaptor ligation step, an aliquot from each address undergoes PCR amplification in order to regenerate a template precursor for the next sequencing cycle.
  • the appropriate primer sets and PCR mix are applied and the array undergoes a number of incubations.
  • the device 20 has a heated stage with a Peltier cooler to accurately and quickly cycle the array through the required amplification regimen, or the array may pass to a separate processing chamber, e.g. an air oven thermal cycler of conventional type, for PCR amplification as illustrated on the bottom of the diagram.
  • new template-precursors are regenerated by PCR amplification, bound to magnetic streptavidin, magnetically pelleted, washed, and cut with FokI, generating a new set of templates corresponding to the previous set of templates but with each strand shortened by nine nucleotides at that end when compared to the prior corresponding template.
  • the automated device may be operated to retain only a small amount of each PCR product for subsequent steps. This can be done by using a streptavidin coated manifold as reported in Lagerkvist A, J Stewart, M Lagerstrom-Fermer, U Landegren. Manifold sequencing. Efficient processing of large sets of sequencing reactions. Proc. Natl. Acad. Sci. USA 1994; 91:2245-2249 and inserting the manifold into the amplification mixture to bind a small proportion of the biotinylated PCR products.
  • cleavable linkage is employed for a portion, e.g. a large fraction, of the linkages used to attach the ligated DNA to the solid support or matrix. Cleavage then releases only the cleavably-bound DNA, permitting removal of a controlled portion of the DNA products.
  • the PCR process may also be controlled by rendering much of the DNA product inaccessible to primer anealing and extension, for example by binding the DNA to a non-dispersible solid matrix or by pelleting a dispersible matrix. This takes advantage of the observation that immobilization of a nucleic acid component during PCR amplification reduces the efficiency of DNA amplification during solid phase PCR. Kohsaka H, D A Carson. Solid Phase Polymerase chain reaction. Journal of Clinical Laboratory Analysis 1994; 8:452-455.
  • This processing obstacle imposed by pre-existing FokI recognition domains may be addressed by hemi-methylating these recognition domains.
  • the methods described in FIGS. 1 and 3 do not provide for the hemi-methylation of those FokI recognition domains that lie outside the adaptor encoded domain.
  • Prior studies such as Looney M C, L S Moran, W E Jack, G R Feehery, J S Benner, B E Slatko, G G Wilson. Nucleotide sequence of the Fok I restriction - modification system: Separate strand - specificity domains in the methyltransferase. Gene 1989; 80:193-208 have shown that hemimethylation of the FokI recognition domain prevents cutting from being mediated by these domains.
  • the thermal cycler can be a Peltier heater-cooler device built into the stage, a set of fixed temperature plates or baths which are successively placed in thermal contact with the chips, or an air oven (see, for example, Meier-Ewert S, E Maier, A Ahmadi, J Curtis, H Lehrach. An automated approach to generating expressed sequence catalogues. Nature 1993; 361:375-376; Drmanac S, R Drmanac. Processing of cDNA and genomic kilobase-size clones for massive screening, mapping, and sequencing by hybridization. BioTechniques 1994; 17: 328-336; Wilding P, M A Shoffner, L J Kricka. PCR in a silicon microstructure.
  • the PCR-based method of Padgett and Sorge has the advantage of allowing the simultaneous exponential amplification of the product of interest along with hemi-methylation of the internal recognition domains. This is accomplished by amplification with a methylated nucleotide that does not lie within the sequence antisense to the recognition domain sequence in the amplifying primer, and can be carried out using ligated adaptors and amplifying primers that vary during each cycle (or every several cycles) as described. In this case, however, the 3′ end of each amplifying primer must encode at least a portion of the restriction endonuclease recognition domain of the class-IIS restriction endonuclease used to trim the DNA segment.
  • any of the above strategies for methylating internal recognition domains can be carried following in vitro amplification of the product of interest, and such prior in vitro amplification could occur through PCR or a related method, such as strand displacement amplification (Walker G T, M S Fraiser, J L Schram, M C Little, J G Nadeau, D P Malinowski Nucleic Acids Research 1992; 20:1691-1696).
  • Such prior DNA amplification in vitro need not have a portion of the recognition domain incorporated into any of the amplifying primers, allowingaki specificity during product regeneration.
  • Each radiolabeled adaptor was added to 25 ⁇ l of the non-radiolabeled adaptors with the other three 5′ ends. This resulted in four adaptor #1 mixes, each with one radiolabeled adaptor and the remaining three non-radiolabeled adaptors. Using four ligation mixtures allows one to sequence nucleotides using a single label and a simple detection apparatus (e.g. a scintillation counter).
  • the steps were identical to the second sequencing cycle, except that the adaptor set used for adaptor ligation was adaptor set #1, and the upper strand of sequencing adaptor set #1 was used as a PCR primer instead of the upper strand of sequencing adaptor set #2.
  • Adaptor set #2 was made the same way as adaptor set #1, except that the four oligonucleotides for the lower strands of the adaptors were:
  • the steps were identical to the first sequencing cycle, except that the adaptor set used for adaptor ligation was adaptor set #2, and the upper strand of sequencing adaptor set #2 was used as a PCR primer instead of the upper strand of sequencing adaptor set #1.
  • the FokI recognition domain is positioned in each ligated adaptor so that one nucleotide was sequenced at 9 nucleotide intervals.
  • the scintillation counts for each of the four adaptors at each sequencing interval is shown below. The highest counts are in bold type.
  • the second adaptor set did not label as efficiently as the first adaptor set. Counts for the correct nucleotide were >12 fold greater than background (counts for any other nucleotide) in the first three cycles. Counts for the correct nucleotide were dominant for cycles 4 and 5, but were less than 2-fold over background.
  • Adaptor set #1 (upper strands of this adaptor set are shown in the box below) was generated as follows: 4.0 ⁇ l of the upper strand of the four adaptors (100 pmole/ ⁇ l) were added, in four separate reactions (one for each oligonucleotide) to 5.0 ⁇ l H 2 O, 16.0 ⁇ l 10 ⁇ Polynucleotide Kinase buffer (700 mM Tris-HCl (pH 7.6), 100 mM MgCl 2 , 50 mM dithiothreitol), 10.0 ⁇ l T 4 Polynucleotide Kinase (10U/ ⁇ l; New England BioLabs, Beverly Mass.) and 125.0 ⁇ l [ 32 P]ATP (2.0 ⁇ Ci/ ⁇ l).
  • the oligonucleotide for the lower strand of the adaptors was:
  • This product was mixed with 460 ⁇ l of washed magnetic streptavidin beads (140 ⁇ l Dynabeads washed and then suspended in 2 ⁇ binding-wash buffer following the manufacturer's instructions), incubated for 1 hour at room temperature (23° C.) with mixing to disperse the magnetic beads, magnetically pelleted (Dynal Magnetic Pellet Concentrator-E), washed three times in binding-wash buffer, and resuspended in 50 ⁇ l TE.
  • the BseRI recognition domain is positioned in each ligated adaptor so that one nucleotide was sequenced at 8 nucleotide intervals.
  • the initial template precursor is shown below, along with its BseRI recognition domain (bold type). Underlined sequences are the original amplifying primers (Primer A and Primer B).
  • the cut sites for this recognition domain, as well as subsequent cut sites directed by ligated adaptors, are shown by dissecting lines. Cleavage generates a single-strand overhang that constitutes a template, and the nucleotide sequenced at each interval is shown by a numbered asterisk, the number identifying the sequencing cycle for sequencing the nucleotide.
  • a FokI based protocol was used to generate a series of templates separated by intervals of nine nucleotides.
  • the initial template precursor was the identical 93 bp PCR product that was used as the initial template precursor in Example 1.
  • each unique PCR amplifying primer used during the sequencing cycles was identical to the upper strand of the previously used adaptor.
  • sequencing was simulated by the incorporation of a ddNTP into the template during five sequencing cycles, and successful trimming of the template was confirmed by acrylamide gel resolution of the PCR products constituting the template precursors during each simulated sequencing cycle.
  • the template was trimmed as predicted over the five sequencing cycles. The details are given below:
  • Adaptor #2 was made the same way as adaptor set #1, except that the oligonucleotide for the lower strand of adaptor #2 was:
  • This product was digested with 1.0 ⁇ l FokI (3U/ ⁇ l) with mixing every 15 minutes in the 1 ⁇ restriction endonuclease buffer in a total volume of 100 ⁇ l at 37° C. for 1 hour, magnetically pelleted, washed three times in binding-wash buffer, and resuspended in 25 ⁇ l H 2 O.
  • the steps were identical to the first sequencing cycle, except that the adaptor used for adaptor ligation was adaptor #2, and the upper strand of adaptor #2 was used as a PCR primer instead of the upper strand of adaptor #1.

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PCT/US1999/004883 WO1999045153A2 (fr) 1998-03-05 1999-03-04 Methode iterative et regenerative de sequencage de l'adn
AU30695/99A AU3069599A (en) 1998-03-05 1999-03-04 An iterative and regenerative dna sequencing method
US09/837,621 US20030044784A1 (en) 1996-11-01 2001-04-17 Iterative and regenerative DNA sequencing method
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US20030092005A1 (en) * 1999-05-19 2003-05-15 Levene Michael J. Optical field enhancement
US20030099956A1 (en) * 2001-09-28 2003-05-29 Brian Ward Recombinant DNA processes using a dNTP mixture containing modified nucleotides
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US20030224439A1 (en) * 2002-05-31 2003-12-04 Mike Lafferty Multiplexed systems for nucleic acid sequencing
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US7476734B2 (en) 2005-12-06 2009-01-13 Helicos Biosciences Corporation Nucleotide analogs
US7482120B2 (en) 2005-01-28 2009-01-27 Helicos Biosciences Corporation Methods and compositions for improving fidelity in a nucleic acid synthesis reaction
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US7645596B2 (en) 1998-05-01 2010-01-12 Arizona Board Of Regents Method of determining the nucleotide sequence of oligonucleotides and DNA molecules
WO2010011944A2 (fr) 2008-07-25 2010-01-28 Wagner Richard W Procédés de criblage de protéines
US7666593B2 (en) 2005-08-26 2010-02-23 Helicos Biosciences Corporation Single molecule sequencing of captured nucleic acids
WO2011011823A1 (fr) * 2009-07-29 2011-02-03 Pyrobett Pte Ltd Procédé et appareil pour conduire un essai
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